The hypothesis to be tested is that an optimal surfactant must have a relatively large fraction of disordered, fluid phase in bilayers to promote a low solution viscosity and rapid adsorption to the interface. This composition must then be refined to produce a higher solid phase fraction in monolayers to promote a surface viscosity large enough to promote high monolayer collapse pressures (low minimum surface tensions) as well as retain surfactant in the alveolus. These mutually exclusive requirements require that a surfactant "reservoir" adjacent to the air-water interface form that is fed simultaneously by monolayer collapse structures and bilayer structures adsorbed from solution. Theoretical predictions of collapse structures, the rates of adsorption to the interface, and the adhesion of surfactant to the interface relate the 3-dimensional (3D) structures to parameters including surface shear viscosity, elasticity, bending modulus, surface pressure etc. These parameters depend on lipid and protein composition, which determines fluid-solid coexistence. Cholesterol also plays an important role in determining surface viscosity and elasticity. To test these theories and to use them to define replacement surfactant composition, confocal microscopy will be developed to investigate the surfactant interface and the 10 - 100 microns immediately adjacent to the interface. In addition, surface viscosity, Langmuir isotherms, fluorescence optical microscopy, atomic force microscopy, and freeze-fracture electron microscopy will be used to relate surfactant monolayer and bilayer properties to surfactant composition. A family of synthetic peptides based on the known amino acid sequences of the hydrophobic surfactant proteins SP-B and SP-C will be designed and synthesized to characterize the effects of sequence, secondary structure and tertiary folding on surfactant function. Peptides will include functional domains (e.g., amphipathic-helical sequences) of the protein known to associate with lipids and membrane-mimic systems. SP-B peptides will be based on the templated structure of NK-lysin and will include monomer and dimer constructs. SP-C protein constructs will be based on the known NMR solution structure of native pig SP-C and a recombinant full length SP-C, and will also include a new dimer SP-C. These peptides and mutants will be used to identify key amino acids that participate in the secondary structure, tertiary folding and quaternary associations of the peptides with surfactant lipids. The overall goal of these studies is to understand the peptide features necessary to maximize in vivo activity.